FIGURE 5.

Effects of Ptc1 on Thr-210 phosphorylation in the absence of the SNF1 heterotrimer. A, cells of the indicated genotype expressed Snf1(1–309)-myc from pXX7 and carried empty vector (V) or overexpressed HA-Ptc1 or HA-Ptc1D58N from pAR12 or pAR32, respectively. Snf1 Thr-210 phosphorylation was assayed as described in Fig. 1. Similar results were observed in snf1Δ and reg1Δ sit4Δ snf1Δ cells. B, ptc1Δ cells carried vector, overexpressed HA-Ptc1 from pAR12, or expressed HA-Ptc1 from its own promoter from pAR48. Protein extracts were prepared from three independent transformants of each type and subjected to immunoblot analysis with anti-HA. The membrane was reprobed with anti-α-tubulin to control for loading. C, cells of the indicated genotype also carried genomic SNF1–8xmyc, and Thr-210 phosphorylation was assayed as described above. Snf1 protein levels are reduced in the reg1Δ sit4Δ snf4Δ glc3Δ samples, relative to snf4Δ glc3Δ controls. In additional controls, reg1Δ snf4Δ glc3Δ SNF1–8xmyc and sit4Δ snf4Δ glc3Δ SNF1–8xmyc cells exhibited glucose-regulated Thr-210 phosphorylation. H, high glucose; L, low glucose.